We have observed that phospholipase A2 (PLA2) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA2s in these processes. The PLA2-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1 naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca2+-independent PLA2 (iPLA2)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca2+-dependent phospholipoase (cPLA2)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. AA release was measured by isotope dilution mass spectrometry. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA2, (which appears to be constituitively active) and cPLA2 (which appears to be sti mulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA2.